Sampling, microbiological methods and data processing of zoonotic and
other enteropathogenic bacteria
Sampling strategy - animals
Salmonella
Samples from animals were collected according to The Norwegian Salmonella Control Programme for Live Animals. Additional samples were obtained from animals during clinical examinations or necropsies at the National Veterinary Institute. One isolate of each serovar per incident was included for susceptibility testing.
Campylobacter jejuni
As part of the Norwegian action plan against Campylobacter in broilers (www.zoonose.no), cloacal samples from chickens were collected at farm level or at slaughter plants, and samples from fresh broiler products were collected at retail level. One isolate per positive farm or batch of products was included for susceptibility testing.
Sampling strategy - humans
Salmonella, Yersinia enterocolitica and Shigella
All the human isolates were obtained from clinical specimens. One isolate per patient was included for susceptibility testing.
Campylobacter
A total of 250 human isolates were obtained from clinical specimens. Five regional laboratories submitted the first five isolates each month to the National Reference Laboratory for Enteropathogenic Bacteria at the Norwegian Institute of Public Health. One isolate per patient was included for susceptibility testing.
Isolation and identification of bacteria
Isolation and identification of Salmonella from animals were carried out by the National Veterinary Institute according to the Nordic Committee on Food Analyses (NMKL) method number 71. Isolation and identification of Campylobacter jejuni from broilers were carried out by the Municipal Food Control Authorities according to the Nordic Committee on Food Analyses (NMKL) method number 119, with minor modifications.
Isolation and identification of bacteria from humans were performed according to conventional methods described in standard reference literature (e.g. the ASM Manual of Clinical Microbiology, Edwards and Ewings Identification of Enterobacteriaceae). The identification of all isolates from animals and humans were verified at the National Reference Laboratory for Enteropathogenic Bacteria at the Norwegian Institute of Public Health.
Susceptibility testing
The isolates from animals were tested for antimicrobial susceptibility at the National Veterinary Institute. MIC values were obtained using the VetMICTM microdilution method (Dept. of Antibiotics, National Veterinary Institute, Sweden).
The Salmonella, Yersinia and Shigella isolates from humans were tested for antimicrobial susceptibility at the Norwegian Institute of Public Health by an agar disc diffusion test using PDM II agar plates and PDM discs (AB Biodisk, Solna, Sweden). The Campylobacter isolates from humans were tested for antimicrobial susceptibility at the Norwegian Institute of Public Health by using Etest (AB Biodisk).
For animal isolates, microbiological breakpoints were mostly used. However, NCCLS breakpoints were applied when available and appropriate. For human isolates, breakpoints defined by AFA (Norwegian Reference Group on Antibiotic Susceptibility Testing) were applied.
Quality assurance systems
The National Veterinary Institute and the Reference Laboratory for Enteropathogenic Bacteria at the Norwegian Institute of Public Health have a quality assurance system according to the requirements of NS-EN ISO/IEC 17025.
Campylobacter jejuni subsp. jejuni CCUG 33057and CCUG 33560 were used as quality control at the National Veterinary Institute on a weekly basis. The National Veterinary Institute participates in an external quality assurance programme for veterinary pathogens. The programme is organized by the VLQAS (Veterinary Laboratories Agency Quality Assurance Unit, Loughborough, England).
The Norwegian Institute of Public Health participates in the external quality assessment programme for Salmonella organized by Enter-Net.
Data processing
Susceptibility data were recorded and processed in WHONET5, a program developed by the World Health Organization (WHO) for analysis of antimicrobial resistance data (ftp.who.int/data/cds/csreph).
Appendix 5
General considerations
NORM is based upon periodic sampling of bacteria from patients with respiratory tract infections, wound infections, urinary tract infections, or septicemiae. For enteric infections see Appendix 4. 2002 was the third year of surveillance, and all twenty-five laboratories in Norway participated in the surveillance system in addition to the National Institute of Public Health. The surveillance strategy is based on sampling and local testing of bacterial isolates from defined clinical conditions. All laboratories follow the same sampling strategy and use identical criteria for the inclusion of bacterial strains. Only one isolate per patient and infectious episode is included. All bacteria were identified using conventional methods as described in the ASM Manual of Clinical Microbiology (7th ed). The surveillance period started in the beginning of January, and consecutive bacterial isolates were included up to a defined maximum of isolates for each surveillance category. The surveillance categories in 2002 were: E.
coli, Klebsiella spp., Staphylococcus aureus,
Streptococcus pneumoniae and Enterococcus spp. from blood cultures; Streptococcus pyogenes from respiratory tract infections and from wound infections and E. coli from urinary tract infections. Blood culture isolates, respiratory tract isolates and isolates from wound specimens were tested using Etest (AB Biodisk, Solna, Sweden), while isolates from urinary tract infections were examined by a disc diffusion method in accordance with the Norwegian Reference Group on Antibiotic Susceptibility Testing (AFA). All resistance values were recorded either as MICs or mm inhibition zone sizes in order to monitor trends in the occurrence of resistance. Suspected MRSA (S. aureus with oxacillin MIC > 4 mg/L) were to be confirmed by mecA PCR, and suspected VRE (enterococci growing on BHI with 6 mg/L vancomycin) were to be confirmed by PCRs for the van gene complex. A computer program (the NORM program) was used for the registration of patient data, sample data and resistance data. Data were analyzed by WHONET5 with the aid of a special program (NORMlink developed by John Stelling) converting the data base structure of NORM to a single file format. Baclink was subsequently used to convert data to the WHONET format
Blood culture isolates
Consecutive isolates of up to 50 each of E. coli, S. aureus, and pneumococci, up to 25 isolates of Klebsiella spp., and up to 20 isolates of enterococci from January until testing time in September to October were included in the surveillance. All isolates were identified to the species level using conventional bacteriological methods. All isolates were tested using Etest (AB Biodisk, Solna, Sweden). A total of 973 isolates of E. coli, 327 isolates of Klebsiella spp, 726 isolates of S. aureus and 252 isolates of enterococci were tested on PDM agar at 35ºC in ambient air, while the 538 isolates of pneumococci were tested on PDM (AB Biodisk, Solna, Sweden) agar supplemented with 5% lysed horse blood at 35ºC in 5%
CO2. All S. aureus isolates were tested for production of ȕ-lactamase using either the nitrocefin disk, the acidometric agar plate (3.6 mg/L penicillin G and phenol red) or the clover leaf method. All S. aureus isolates were screened for methicillin-resistance using MH agar (Difco) with 4% NaCl and oxacillin 4 mg/L and a spot inoculum of 106 cfu/spot. All enterococci were screened for vancomycin resistance using BHI agar (Difco) and vancomycin 6 mg/L. The following strains were used for quality control: E. coli ATCC 25922, E. faecalis ATCC 29212, S. pneumoniae ATCC 49619, S. aureus ATCC 29213, S. aureus ATCC 43300 (heterogeneous methicillin resistance), and S. aureus CCUG 35600 (homogeneous methicillin resistance)
Respiratory tract isolates
Up to 50 consecutive isolates each of S. pyogenes from patients with respiratory tract infections were collected in each laboratory from January to March. All isolates were kept in a freezer and tested in batch using Etest (AB Biodisk, Solna Sweden). A total number of 568 S. pyogenes were included in the study. S. pyogenes were tested on PDM agar supplemented with 5% lysed horse blood at 35ºC in 5% CO2. The following strain was used for quality control: S. pneumoniae ATCC 49619.
Wound specimens
Up to 50 consecutive isolates of S. pyogenes from patients with wound infections were collected in each laboratory from January to March. All isolates were kept in a freezer and tested in batch using Etest (AB Biodisk, Solna Sweden). A total of 395 S. pyogenes were included in the study. S. pyogenes were tested on PDM agar supplemented with 5% lysed horse blood at 35ºC in 5% CO2. The following strain was used for quality control: S. pneumoniae ATCC 49619.
Urinary tract isolates
Up to 50 consecutive isolates of E. coli from patients with urinary tract infections were collected in each lab during January and February. All isolates were either kept on bench or in a freezer until tested in batch using a disk diffusion method with PDM agar and paper disk (AB Biodisk, Solna Sweden) at 35ºC in ambient air. The study included 1044 E. coli isolates. The following strain was used for quality control: E. coli ATCC 25922.
Mycobacterium tuberculosis
In the year 2002, antimicrobial susceptibility testing of M. tuberculosis was performed at the following institutions: National Institute of Public Health, Oslo, Ullevål University Hospital, Oslo, National Hospital, Oslo, and Haukeland Hospital, Bergen. The majority of isolates were tested using the BACTEC (National Institute of Public Health and Ullevål University Hospital) or MGIT systems (National Hospital). All four laboratories participate in an external quality control program organized by the WHO.